Application of silicate bioactive ceramic material with anti-aging and osteogenesis-promoting properties

Abstract

The invention relates to application of a silicate bioactive ceramic material with anti-aging and osteogenesis promoting properties. The silicate bioactive ceramic material is a zincite material, and the chemical composition of the silicate bioactive ceramic material is Ca2ZnSi2O7. The form of the melilite material is a melilite bracket, a melilite leaching solution or a melilite dispersion solution; the silicate bioactive ceramic material continuously releases Zn < 2 + > and SiO3 < 2-> to synergistically activate an SIRT1-mediated PI3K-AKT signal channel so as to realize cell aging inhibition and bone tissue regeneration; wherein the melilite scaffold is used for preparing an implant material for regeneration of senescent tissues; the melilite leach liquor or the melilite dispersion liquid is used for preparing the anti-aging and osteogenesis-promoting double-effect oral preparation.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and relates to the application of a silicate bioceramic material with dual anti-aging and osteogenic functions in the treatment of age-related bone diseases. Background Technology

[0002] Aging is an inherent property of life, and it is unavoidable, from the replicative aging of single cells to the programmed decline of multicellular organisms. Humanity's long-held desire to combat aging has become a key objective of contemporary biomedicine. Therefore, a deep understanding of the mechanisms of aging and the development of targeted intervention strategies to address age-related pathologies are crucial.

[0003] Modern molecular biology has revealed the core characteristics of cellular senescence: irreversible cell cycle arrest, continuous secretion of senescence-associated phenotypic (SASP) phenotypes, and the resulting imbalance in the bone microenvironment. In the skeletal system, elevated SASP factors in senescent bone tissue impair the proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs), creating a vicious cycle of decreased regenerative capacity, ultimately leading to osteoporosis and delayed fracture healing. Current anti-aging therapies mainly consist of two classes of drugs: one is "senescent cell scavengers," which reduce tissue burden by inducing apoptosis of senescent cells; the other is "SASP modulators," which alleviate bone microenvironment disorders by inhibiting the release of inflammatory factors. However, the potential side effects, limited efficacy, and toxicity of these drugs hinder their clinical application, and they generally lack the ability to promote bone tissue regeneration. In contrast, anti-aging biomaterials, especially bone repair materials (such as reactive oxygen species-regulated hydrogels and targeted delivery functional materials), are developing rapidly, demonstrating the advantage of promoting bone regeneration by regulating stem cells, inflammation, and angiogenesis. However, existing materials still face key bottlenecks: a mismatch between mechanical properties and degradation rates (hydrogels are prone to collapse, while metals are difficult to degrade); insufficient regulation of complex microenvironments such as vascularization, nerve innervation, and multicellular network synergy; and a lack of temporal dynamism in the "one-time release" strategy, making it difficult to adapt to the needs of different stages of bone repair (such as early anti-inflammatory to late-stage mineralization). Currently, anti-aging drugs, such as the combination of dasatinib and quercetin, can clear senescent cells, but they have serious side effects and lack bone regeneration capabilities; traditional bone repair materials, such as β-TCP ceramics, can promote osteogenic differentiation but cannot regulate the aging microenvironment. Therefore, developing material systems that combine anti-aging and bone regeneration functions is crucial for the treatment of age-related bone diseases. Summary of the Invention

[0004] To overcome the shortcomings of existing anti-aging drugs lacking bone regeneration function and bioceramic materials being unable to regulate the aging microenvironment, this invention provides a bifunctional material system based on zinc-doped silicate ceramics. This system simultaneously achieves cell senescence inhibition and bone tissue regeneration through a specific ion release mechanism, and establishes a multidimensional therapeutic platform comprising an implantable scaffold, an injectable suspension, and an oral formulation.

[0005] This invention provides an application of a silicate bioactive ceramic material with both anti-aging and bone-promoting properties. The silicate bioactive ceramic material is zinc feldspar, with the chemical composition Ca2ZnSi2O7. The zinc feldspar material is available in the form of a zinc feldspar scaffold, a zinc feldspar extract, or a zinc feldspar dispersion. The silicate bioactive ceramic material continuously releases Zn... 2+ The zinc feldspar scaffold is used to synergistically activate the SIRT1-mediated PI3K-AKT signaling pathway with SiO32- to inhibit cell senescence and promote bone tissue regeneration; wherein, the zinc feldspar scaffold is used to prepare implantable materials for the regeneration of senescent tissues; and the zinc feldspar extract or zinc feldspar dispersion is used to prepare an oral formulation with dual anti-aging and osteopromoting effects.

[0006] In this invention, the silicate bioactive ceramic material uses pure-phase zinc feldspar (Ca2ZnSi2O7, ZnCS) as the active core, wherein calcium, zinc, and silicon elements are uniformly spatially distributed. The silicate bioactive ceramic material continuously releases zinc (ZnCS) at a concentration of 0.3–12.5 μg / mL. 2+ ) and 5.0–50.0 μg / mL silicon (SiO32-) synergistically activate the SIRT1 / PI3K-AKT signaling pathway, reducing p16 protein expression in senescent bone marrow mesenchymal stem cells and decreasing the proportion of SA-β-gal positive cells, while significantly increasing the expression level of the key osteogenic gene RUNX2, promoting the increase of new bone volume fraction. Ion release is controlled by designing the physical morphology of the material, with the core being the regulation of the contact area between zinc feldspar and the surrounding environment. For the scaffold, the specific surface area is increased by adjusting porosity and connectivity, thereby achieving post-implantation Zn 2+ The long-term, sustained release of SiO32- is suitable for constructing a bone regeneration microenvironment. For the extract, gradient dilution is used to obtain the desired specific concentration of ions, providing controllable and reproducible stimulation conditions for in vitro cell experiments. For the dispersion system, the concentration of released ions is controlled by adjusting the concentration of solid powder in the culture environment; ultimately, the ion concentration is precisely adjusted through the particle concentration in the dispersion.

[0007] Preferably, the silicate bioactive ceramic material releases Zn 2+ The concentration of Zn is 0.3–12.5 μg / mL, and the concentration of SiO32- is 5.0–50.0 μg / mL. In this invention, ion release is controlled by designing the physical morphology of the material, with the core being the regulation of the contact area between zinc feldspar and the surrounding environment. For the scaffold, the specific surface area is increased by adjusting porosity and connectivity, thereby achieving post-implantation Zn... 2+The long-term, sustained release of SiO32- is suitable for constructing a bone regeneration microenvironment. For the extract, gradient dilution is used to obtain the desired specific concentration of ions, providing controllable and reproducible stimulation conditions for in vitro cell experiments. For the dispersion system, the concentration of released ions is controlled by adjusting the concentration of solid powder in the culture environment; ultimately, the ion concentration is precisely adjusted through the particle concentration in the dispersion.

[0008] Preferably, the zinc feldspar material is in the form of a zinc feldspar support; the macroscopic morphology of the zinc feldspar support is a multi-layered mesh support formed by multiple base columns stacked at 45°; preferably, the diameter of a single base column in the zinc feldspar support is 0.4-0.6 mm; the distance between the base columns is 0.8-1.2 mm.

[0009] Preferably, the zinc feldspar support has a three-dimensional interconnected pore structure; the pore size of the three-dimensional interconnected pore structure is 200-800 μm, and the porosity is 30-70%.

[0010] Preferably, the method for preparing the zinc feldspar scaffold includes: (1) Mix zinc feldspar powder with photosensitive resin to obtain photocurable printing paste; (2) Zinc feldspar scaffold blanks were prepared by photopolymerization printing technology; (3) The obtained zinc feldspar support blank is sintered to obtain the zinc feldspar support.

[0011] Preferably, in step (1), the mass ratio of the zinc feldspar powder to the photosensitive resin is (40-50):55.

[0012] Preferably, in step (1), the mixing method is ball milling, the ball milling speed is 400-600 r / h, and the time is 0.5-2 h.

[0013] Preferably, in step (2), the solid content of the photocurable printing paste is 40-50 wt.%.

[0014] Preferably, in step (3), the sintering temperature is 1250–1270°C and the holding time is 3–3.5 h; preferably, the sintering process includes: heating to 200–400°C at a heating rate of 0.5–2°C / min and holding for 1–2 hours to remove moisture from the support; then heating to 600–700°C at a heating rate of 0.2–1°C / min and holding for 2–6 hours to remove polymer components from the support; finally heating to 1250–1270°C at a heating rate of 0.5–2°C / min and holding for 2–6 hours, followed by natural cooling.

[0015] Preferably, the zinc feldspar material is in the form of a zinc feldspar extract; the preparation method of the zinc feldspar extract includes: mixing zinc feldspar powder with a basic culture medium at a ratio of 200 mg / mL to obtain a mixture; after centrifuging the mixture on a shaker, collecting the supernatant and sterilizing it to obtain the zinc feldspar extract.

[0016] Preferably, the particle size of the zinc feldspar powder is 8.0–70 μm; and the basal culture medium is MEM-α medium.

[0017] Preferably, the temperature of the shaker is 37°C and the time is 24 hours.

[0018] Preferably, the concentration of zinc ions in the zinc feldspar extract is 0.3–0.6 μg / mL, and the concentration of silicate ions is 43–50 μg / mL.

[0019] Preferably, the zinc feldspar material is in the form of a zinc feldspar dispersion; the preparation method of the zinc feldspar dispersion includes: mixing sterilized zinc feldspar powder with a basic culture medium at a ratio of 25-500 μg / mL to obtain the zinc feldspar dispersion.

[0020] Preferably, the zinc feldspar powder is disinfected by ultraviolet light for 12–48 hours.

[0021] In this invention, the zinc feldspar scaffold is used to prepare implantable materials for the regeneration of aging tissues. When implanted, the active ion release cycle exceeds 28 days, and after 12 weeks, the bone mineralization density in the defect area recovers to more than 80% of that of normal bone. The zinc feldspar extract or zinc feldspar dispersion is used to prepare an oral preparation with dual effects of anti-aging and osteopromoting effects. When administered at a dose of 0.4–0.8 mL daily, after 8 weeks of continuous administration, the number of trabeculae in the osteoporosis model increases significantly, which can effectively delay bone loss after ovariectomy.

[0022] The inventive breakthrough of this invention stems from its dual synergistic molecular-structural mechanism. At the molecular level, zinc ions enhance the deacetylase activity of the SIRT1 protein by stabilizing its catalytic structure, effectively inhibiting the p53 phosphorylation process; while silicate ions activate AMPK / NAD... + Metabolic pathways enhance SIRT1 transcriptional expression. The synergistic effect of these two mechanisms drives increased β-catenin nuclear translocation efficiency and simultaneously blocks PPARG-mediated adipogenic differentiation. Structurally, the scaffold employs a three-dimensional perforated configuration with pore sizes strictly controlled within the range of 200–800 μm and a porosity of 30–70%, resulting in an active ion release cycle exceeding 28 days after implantation. Oral administration at doses of 0.4–0.8 mL daily significantly increased the number of trabeculae in the osteoporosis model after 8 weeks.

[0023] Beneficial effects: The silicate bioactive ceramic material (ZnCS) provided in this invention, possessing both anti-aging and osteogenic properties, exhibits dual bioactivity in vitro in three forms (porous scaffold, extract (or ion extract), and dispersion (or particulate suspension), simultaneously delaying aging and promoting osteogenic regeneration. Compared to traditional β-TCP bioceramics, ZnCS demonstrates better ability to remodel the aging microenvironment and significantly accelerates bone regeneration in osteoporotic defects. Furthermore, ZnCS has been shown to enable effective oral treatment, reducing osteoporosis-related bone loss. This silica-based bioceramic, ZnCS, successfully combines anti-aging and osteoinductive properties, expanding the functional range of silica-based materials and enriching the theoretical framework of bioceramic science. Attached Figure Description

[0024] Figure 1 Optical photograph of the zinc feldspar scaffold after 3D printing and sintering; Figure 2 A is an optical photograph of the 3D-printed and sintered β-tricalcium phosphate scaffold. Figure 2 B is an optical photograph of the 3D-printed and sintered zirconia support. Figure 3 To verify the dual biological activities of ZnCS scaffolds in anti-aging and osteopromoting processes, the following steps were performed: AE: Real-time quantitative PCR (qPCR) was used to detect the expression levels of aging-related genes (p16(A), p21(B), p53(C), IL-6(D), IL-8(E)) in each group (Normal, ZnCS-Normal, Doxorubicin-induced aging group (Dox), and scaffold-treated aging group (TCP-Dox, ZrO2-Dox, and ZnCS-Dox)); FI: qPCR was used to detect the expression levels of osteogenic genes (ALP(F), BMP2(G), RUNX2(H), OPN(I)) in each group (Normal, ZnCS-Normal, Doxorubicin-induced aging group (Dox), and scaffold-treated aging group (TCP-Dox, ZrO2-Dox, and ZnCS-Dox)). Figure 4To verify the dual bioactivity of ZnCS ion extract in anti-aging and osteopromoting effects; AE was the detection of the expression levels of aging-related genes (p16(A), p21(B), p53(C), IL-6(D), IL-8(E)) in each group (normal group, doxorubicin-induced aging group, and 1 / 8 concentration ZnCS extract treatment group (1 / 8ZnCS)) by real-time quantitative PCR (qPCR); FI was the detection of the expression levels of osteogenic genes (ALP(F), BMP2(G), RUNX2(H), OPN(I)) in each group (normal group, doxorubicin-induced aging group, and 1 / 8 concentration ZnCS extract treatment group (1 / 8ZnCS)) by qPCR. Figure 5 The interaction between ZnCS particle dispersion and cells is shown; where A is a transmission electron microscope (TEM) ultramicrograph of ZnCS particles internalized in BMSCs (red arrow indicates endocytosed particles); B is a fluorescent ZnCS particle (green) and lysosomal compartment (…). Co-localization analysis of red (C); immunofluorescence localization analysis of fluorescent ZnCS particles (green) and cytoskeleton (FITC, red); Figure 6 ZnCS particle dispersion possesses dual bioactivity of anti-aging and osteopromoting effects; AE represents the expression levels of aging-related genes (p16(A), p21(B), p53(C), IL-6(D), IL-8(E)) in each group (normal group, doxorubicin-induced aging group, and 50μg / mL ZnCS particle suspension treatment group); FI represents the expression levels of osteogenic genes (ALP(F), BMP2(F), RUNX2(H), OPN(I)) in each group (normal group, doxorubicin-induced aging group, and 50μg / mL ZnCS particle suspension treatment group). Figure 7 ZnCS porous scaffold implantation in osteoporotic rabbits with femoral defects effectively promotes bone regeneration. A shows three-dimensional reconstructed Micro-CT images of new bone formation (green) and scaffold integration (red) in the sham surgery group (Sham), the ovariectomy blank defect group (OVX-Blank), the ovariectomy TCP scaffold implantation group (OVX-TCP), and the ovariectomy ZnCS scaffold implantation group (OVX-ZnCS). B shows quantitative bone parameters of the newly formed bone (quantitative statistical analysis of bone mineral density (BMD), C shows quantitative statistical analysis of bone volume fraction of newly formed bone (BV / TV), and D shows quantitative statistical analysis of trabecular bone thickness (Tb.Th.) of newly formed bone). Figure 8 Biological effects of ZnCS porous scaffold implanted in osteoporotic rabbit femoral defects; where A is the immunofluorescence localization map of the aging marker p16; B is the immunofluorescence localization map of osteogenic protein RUNX2; C is the quantitative analysis of p16 protein expression in bone tissue; D is the quantitative analysis of RUNX2 protein expression in bone tissue; Figure 9 Figure 1 shows the experimental results of oral ZnCS powder effectively delaying bone loss. In this figure, A is a three-dimensional Micro-CT reconstructed image of the femoral trabecular structure; B is the quantitative analysis of bone morphometrics in each group (sham surgery group, oophorectomy group, and oral ZnCS powder treatment group (OVX+ZnCS)) including bone volume fraction (BV / TV), number of trabeculae (Tb.N), and trabecular separation (Tb.Sp)). Figure 10 The in vivo biological effects of oral ZnCS powder are shown in Figure 1. A is the spatial localization map of the aging marker p16; B is the spatial localization map of the osteogenic regulator RUNX2; C is the quantitative analysis of p16 protein expression in bone tissue; and D is the quantitative analysis of RUNX2 protein expression in bone tissue. Detailed Implementation

[0025] To further illustrate the invention's content, features, and practical effects, the invention will be described in detail below with reference to embodiments. It should be noted that the modification methods of the invention are not limited to these specific implementation methods. Equivalent substitutions and modifications made by those skilled in the art based on their reading of the invention's content, without departing from the spirit and essence of the invention, are also within the scope of protection claimed by this invention.

[0026] This invention provides an application of a silicate bioactive ceramic material with both anti-aging and bone-promoting properties. The silicate bioactive ceramic material is zinc feldspar, with the chemical composition Ca2ZnSi2O7. The zinc feldspar material is available in the form of a zinc feldspar scaffold, a zinc feldspar extract, or a zinc feldspar dispersion. The silicate bioactive ceramic material continuously releases Zn... 2+This invention utilizes SiO32- to synergistically activate the SIRT1-mediated PI3K-AKT signaling pathway, thereby inhibiting cellular senescence and promoting bone tissue regeneration. The zinc feldspar scaffold is used to prepare implantable materials for regenerating senescent tissues. The zinc feldspar extract or dispersion is used to prepare an oral formulation with both anti-aging and osteogenic effects. This invention designs and optimizes different forms of zinc feldspar materials, including zinc feldspar scaffolds, zinc feldspar extracts, and zinc feldspar dispersions, all of which can effectively delay the senescence process of bone marrow mesenchymal stem cells (BMSCs) and effectively promote their osteogenic differentiation, demonstrating their feasibility as implantable materials for regenerating senescent bone tissue. Preferably, the zinc feldspar scaffold can significantly accelerate bone regeneration in osteoporotic bone defects. Furthermore, the silicate-active bioceramic material can also be used as an oral therapeutic agent to effectively alleviate bone loss caused by osteoporosis. The silicate bioactive ceramic based on zinc feldspar provided by this invention effectively integrates anti-aging and osteogenic induction properties, expands the functional range of silicon-based materials, and can promote the regeneration of aging bone tissue or delay bone loss in aging bone tissue in different forms, thus protecting the bone health of the elderly.

[0027] This invention is the first to propose the concept of "Silicate Anti-Aging (SAS)," which realizes the dual biological functions of ZnCS through three forms: porous scaffold, extract (or ionic extract), and dispersion (or particulate suspension). (1) Anti-aging: Downregulates p16 / p21 / p53 aging genes and SASP factors such as IL-6 / IL-8. (2) Bone regeneration: Activation of osteogenic gene expression such as RUNX2 / OPN / BMP2. Specifically, this invention utilizes ZnCS materials to continuously release Zn 2+ Together with SiO32-, it synergistically activates the core regulatory factor of SIRT1, Zn 2+ By enhancing SIRT1 enzyme activity, SiO32- promotes SIRT1 expression, thus jointly activating SIRT1 function. Activated SIRT1 further inhibits the p53-dependent aging pathway and regulates downstream targets through deacetylation modification. ZnCS materials, on the one hand, delay cellular senescence and reduce aging-related phenotypes by inhibiting the p53 signaling pathway; on the other hand, they upregulate osteogenic gene expression by activating the PI3K-Akt signaling pathway while inhibiting adipogenic differentiation. This allows for the simultaneous and efficient anti-aging and bone regeneration promotion within the aging microenvironment, overcoming the limitation of existing anti-aging drugs that cannot synergistically promote bone growth.

[0028] When the zinc feldspar material is in the form of a zinc feldspar scaffold, the following exemplarily illustrates the preparation method of the zinc feldspar scaffold.

[0029] Preparation of the initial slurry. 33–56 wt% of zinc feldspar powder is mixed with photosensitive resin and ball-milled at 400–600 r / h for 0.5–2 h to obtain the initial slurry. The photosensitive resin is selected from at least one of epoxy resin, acrylate, epoxy acrylate, and washable E-Sheng W100; in some embodiments, the photosensitive resin is washable E-Sheng W100.

[0030] Preparation of photocurable printing paste. The remaining zinc feldspar powder was mixed with the obtained initial paste at a mass ratio of (15–35):(70–80), and ball-milled at a speed of 400–600 r / h for 1–2 h to obtain the photocurable printing paste. The solid content of the photocurable printing paste was 40–50 wt.%.

[0031] Printing of the support frame blank. The support frame model is designed using 3D modeling software (such as 3ds Max). The following parameters can be selected: the macroscopic shape of the support frame is a cylinder with a diameter of 6mm and a height of 6mm; the helical angle between the upper and lower base columns is 45°; the diameter of a single base column is 0.8mm; the distance between each layer of base columns is 0.6mm; the number of base columns in the horizontal direction is 6; and the overlap height between the upper and lower base columns in the vertical direction is 0.1–0.2mm. Then, the designed support frame model is imported into slicing software for slicing, with a slice thickness set to 50μm. The slices are then imported into a photopolymer 3D printer and printed using printing paste to obtain the support frame blank.

[0032] Sintering. The obtained support blank is calcined at 1250-1270℃ for 3-3.5h to obtain the zinc feldspar support.

[0033] When the zinc feldspar material is in the form of a zinc feldspar extract, the following exemplarily illustrates the preparation method of the zinc feldspar extract.

[0034] Zinc feldspar powder was mixed with basal culture medium at a ratio of 200 mg / mL to obtain a mixture. The mixture was placed in a shaker at 37°C for 24 h. The mixture was centrifuged, and the supernatant was collected and sterilized to obtain the zinc feldspar extract. The particle size of the zinc feldspar powder was 8.0–70 μm. The basal culture medium was MEM-α medium. The concentration of the zinc feldspar extract was 200 mg / mL. The concentration of zinc ions in the zinc feldspar extract was 0.3–0.6 μg / mL, and the concentration of silicate ions was 43–50 μg / mL.

[0035] When the zinc feldspar material is in the form of a zinc feldspar dispersion, the following exemplarily illustrates the preparation method of the zinc feldspar dispersion.

[0036] Zinc feldspar powder was sterilized under ultraviolet light for 12–48 h, and then mixed with basal culture medium at a ratio of 25–500 μg / mL to obtain the zinc feldspar dispersion.

[0037] The zinc feldspar (ZnCS) material provided in this invention, in three forms—porous scaffold, ion release, and particle contact—can stably achieve the dual biological function of delaying BMSC aging and maintaining osteogenic potential. This not only verifies that silicon-based bioceramic materials (represented by ZnCS) can achieve osteogenic potential through ion regulation (Zn... 2+ The innovative concept of using SiO32- and physical interactions to intervene in stem cell aging has laid a scientific foundation for its application in the field of anti-aging bone regeneration.

[0038] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0039] Example 1 The preparation process of the zinc feldspar scaffold in Example 1 includes: (1) Weigh 20g of zinc feldspar powder and 55g of Yisheng W100 water-washed photosensitive resin respectively, and ball mill them at 500r / h for 2h to obtain the initial slurry. (2) Weigh 25g of zinc feldspar powder and 75g of initial slurry prepared in step (1), and ball mill them together at 500r / h for 2h to obtain printing slurry; (3) Zinc feldspar scaffold blanks were prepared by photopolymerization printing technology; (4) The obtained zinc chromate scaffold blank was sintered at 1250℃ for 3h to obtain zinc chromate scaffold (ZnCS Scaffold); (5) The obtained zinc feldspar scaffold was ultrasonically cleaned and sterilized under high temperature and high pressure for subsequent cell experiments.

[0040] Example 2 The preparation process of the zinc feldspar extract in Example 2 includes: mixing zinc feldspar powder with basal culture medium at a ratio of 200 mg / mL, placing it in a shaker at 37°C for 24 h, centrifuging the mixture, collecting the supernatant, sterilizing it through a filter membrane to obtain the zinc feldspar extract stock solution (concentration of 200 mg / mL), and then diluting the zinc feldspar extract stock solution with basal culture medium to obtain the zinc feldspar extract; the concentration of the zinc feldspar extract is the zinc feldspar extract stock solution diluted 1 / 8 times.

[0041] Example 3 The preparation process of the zinc feldspar dispersion in Example 3 includes: sterilizing the zinc feldspar powder with ultraviolet light for 24 hours; mixing the sterilized zinc feldspar powder with the basic culture medium at a ratio of 50 μg / mL to obtain the zinc feldspar dispersion; the concentration of the zinc feldspar dispersion is 50 μg / mL.

[0042] Example 4 Healthy BMSCs were seeded onto the ZnCS scaffold prepared in Example 1 and allowed to adhere overnight. The culture medium was then replaced with MEM-α serum-free medium containing 0.25 μM doxorubicin. After 48 hours of treatment, the culture medium was replaced with complete medium containing 10% FBS and cultured for another 2-6 days. The aging process of the BMSCs was then detected. The BMSCs treated in Example 4 were named the ZnCS-Dox group.

[0043] Example 5 After seeding healthy BMSCs into blank well plates, they were allowed to stand overnight to allow them to adhere. The culture medium was then replaced with MEM-α serum-free medium containing 0.25 μM doxorubicin and the ZnCS extract prepared in Example 2. After 48 hours of treatment, the culture medium was replaced with complete medium containing 10% FBS and a 1 / 8 dilution of ZnCS extract. The cells were cultured for another 2–6 days, and samples were collected to assess the aging process of the BMSCs. The BMSCs treated in Example 5 were designated as the 1 / 8 ZnCS group.

[0044] Example 6 Healthy BMSCs were seeded onto blank plates and allowed to adhere overnight. The culture medium was then replaced with MEM-α serum-free medium containing 0.25 μM doxorubicin and 50 μg / mL ZnCS dispersion. After 48 hours of treatment, the medium was replaced with complete medium containing 10% FBS and 50 μg / mL ZnCS dispersion. The culture was continued for 2–6 days, and samples were collected to assess the aging process of the BMSCs. The BMSCs treated in Example 6 were designated as the 50 μg / mL group.

[0045] Example 7 The process of establishing the osteoporosis rabbit animal model in Example 7 includes: 3-month-old female New Zealand white rabbits were fasted for 12 hours, and then anesthetized with an intravenous injection of 3% sodium pentobarbital (30 mg / kg). Under sterile conditions with full skin exposure, the rabbits were fixed in a prone position on the operating table, and the skin was disinfected with povidone-iodine solution. Ovariectomy was performed dorsally.

[0046] Example 8 The process of establishing the osteoporotic rabbit femoral condyle defect model in Example 8 included the following steps: After the successful establishment of the osteoporotic rabbit animal model in Example 7, the rabbits were anesthetized by intravenous injection of 3% sodium pentobarbital (30 mg / kg). Under sterile conditions with full skin exposure, the rabbits were fixed in a lateral decubitus position on the operating table, and the skin was disinfected with povidone-iodine solution. After exposing the femoral condyle, a femoral condyle defect with H=6 mm and Ф=6 mm was created using a bone drill. Subsequently, a ZnCS scaffold was implanted into the rabbit's femoral defect, and finally, the muscles and skin were sutured one by one. The rabbits treated in Example 8 were named the OVX-ZnCS group.

[0047] Example 9 One week after bilateral ovariectomy, 8-week-old female C57BL / 6 mice were treated orally with ZnCS dispersion. ZnCS was administered via gavage in the form of a saline suspension, with a volume of 0.4–0.6 mL. The solid content of the ZnCS saline suspension was 12–16 mg / mL. This was done once daily for 8 weeks. The mice treated in Example 9 were designated as the OVX@ZnCS group.

[0048] Comparative Example 1 After inoculating healthy BMSCs onto blank well plates, they were left to stand overnight to allow them to adhere. The culture medium was then replaced with complete medium containing 10% FBS, and the plates were cultured for another 4-8 days. The BMSCs treated in Comparative Example 1 were named the Normal group.

[0049] Comparative Example 2 After inoculating healthy BMSCs onto zinc feldspar scaffolds, they were allowed to stand overnight to allow them to adhere. The culture medium was then replaced with complete medium containing 10% FBS, and the cells were cultured for another 4-8 days. The BMSCs treated in Comparative Example 2 were named the ZnCS-Normal group.

[0050] Comparative Example 3 After inoculating healthy BMSCs into blank well plates, they were allowed to adhere overnight. The medium was then replaced with serum-free MEM-α medium containing 0.25 μM doxorubicin and treated for 48 hours. Subsequently, the medium was replaced with complete medium containing 10% FBS and cultured for another 2–6 days. The BMSCs treated in this comparative example 3 were named the Dox group.

[0051] Comparative Example 4 (1) Preparation of β-TCP scaffold: First, weigh 20g of β-TCP powder and 55g of Yisheng W100 water-washable photosensitive resin, and ball mill them together at 500r / h for 2h to obtain an initial slurry; then, mix the remaining (25g) of β-TCP powder with 75g of the initial slurry by ball milling at 500r / h for 2h to obtain a printing slurry; prepare β-TCP scaffold blanks by photopolymerization printing technology; sinter the obtained β-TCP scaffold blanks at 1150℃ for 3h to obtain β-TCP scaffolds (β-TCP Scaffolds); and sterilize the obtained β-TCP scaffolds by ultrasonic cleaning and high temperature and high pressure for subsequent cell experiments. (2) Preparation of ZrO2 scaffold: First, weigh 20g of ZrO2 powder and 55g of Yisheng W100 water-washable photosensitive resin, and ball mill them together at 500r / h for 2h to obtain an initial slurry; then, ball mill the remaining (25g) of ZrO2 powder and 75g of the initial slurry together at 500r / h for 2h to obtain a printing slurry; prepare ZrO2 scaffold blanks by photopolymerization printing technology; sinter the obtained ZrO2 scaffold blanks at 1350℃ for 3h to obtain ZrO2 scaffolds (ZrO2 Scaffolds); and sterilize the obtained ZrO2 scaffolds by ultrasonic cleaning and high temperature and high pressure for subsequent cell experiments. (3) Healthy BMSCs were seeded onto β-TCP scaffolds and ZrO2 scaffolds respectively, and allowed to adhere overnight. The culture medium was changed to MEM-α serum-free medium containing 0.25 μM doxorubicin. After 48 hours of treatment, the culture medium was changed to complete medium containing 10% FBS and cultured for another 2-6 days. The BMSCs treated in Comparative Example 4 were named β-TCP-Dox group and ZrO2-Dox group respectively.

[0052] Comparative Example 5 Healthy 3-month-old female New Zealand white rabbits were anesthetized by intravenous injection of 3% sodium pentobarbital (30 mg / kg). Under sterile conditions with full skin exposure, the rabbits were fixed in a lateral decubitus position on the operating table, and the skin was disinfected with povidone-iodine solution. After exposing the femoral condyle, a femoral condyle defect with H=6 mm and Ф=6 mm was created using a bone drill, and the muscle and skin were sutured directly one by one. The rabbits treated in Comparative Example 5 were named the Sham-Blank group.

[0053] Comparative Example 6 The successfully established osteoporotic rabbits were anesthetized by intravenous injection of 3% sodium pentobarbital (30 mg / kg). Under sterile conditions with full skin exposure, the rabbits were fixed in a lateral decubitus position on the operating table, and the skin was disinfected with povidone-iodine solution. After exposing the femoral condyle, a femoral condyle defect with H=6 mm and Ф=6 mm was created using a bone drill. Subsequently, a β-TCP scaffold was implanted into the bone defect site, or no scaffold material was implanted. Finally, the muscles and skin were sutured one by one. The rabbits treated in Comparative Example 6 were named the OVX-TCP group and the OVX-Blank group, respectively.

[0054] Comparative Example 7 Eight-week-old female C57BL / 6 mice underwent bilateral ovariectomy or sham surgery. One week later, they were given oral saline orally or by gavage at a volume of 0.4–0.6 mL once daily for 8 weeks. The mice treated in Comparative Example 7 were named the OVX group and the Sham group, respectively.

[0055] Inductively coupled plasma atomic emission spectrometry was used to determine the Zn released from the zinc chalcopyrite scaffold, zinc chalcopyrite extract, and zinc chalcopyrite dispersion. 2+ The concentrations of SiO32- and SiO32- are shown in Table 1.

[0056] Table 1: Example <![CDATA[Zn 2+ / μg / mL]]> <![CDATA[SiO32- / μg / mL]]> Example 1 5.00～12.50 5.50～15.00 Example 2 0.30～0.60 43.00～50.00 Example 3 4.00～6.00 5.50～8.50 .

[0057] Figure 1 , Figure 2 These are optical photographs of three 3D-printed and sintered scaffolds at different magnifications. As shown in the figures, the three scaffolds retain an interconnected pore structure after 3D printing and sintering. The zinc feldspar scaffold has a pore size of 200–800 μm and a porosity of approximately 30–70%. The β-TCP scaffold has a pore size of 300–800 μm and a porosity of approximately 30–70%. The ZrO2 scaffold has a pore size of 300–800 μm and a porosity of approximately 30–70%.

[0058] Senescence process and osteogenic potential of BMSCs under the action of zinc feldspar materials

[0059] ZnCS porous scaffolds were successfully fabricated using photopolymerization 3D printing technology, with bioactive ceramic β-TCP and inert ceramic ZrO2 serving as control groups. Experiments showed that the ZnCS scaffold significantly promoted the spreading morphology and proliferative activity of normal bone marrow mesenchymal stem cells (BMSCs) (superior to the control group on day 5 of culture). In a doxorubicin (Dox)-induced senescent BMSCs model, the ZnCS scaffold group (ZnCS-Dox) not only significantly enhanced the proliferative capacity of senescent cells but also effectively downregulated the expression of cell cycle arrest genes (p16, p21, p53) and aging-related secretory phenotypic factors (IL-6, IL-8). Notably, the ZnCS scaffold could reverse the decline in osteogenic differentiation capacity caused by aging: compared with the control group, the ZnCS-Dox group significantly upregulated the gene expression levels of early osteogenic markers (ALP, BMP2, RUNX2) and late markers (OPN), indicating that it has a key function in maintaining the osteogenic differentiation potential of senescent BMSCs (e.g., ...). Figure 3 (As shown).

[0060] To elucidate the mechanism of action of ZnCS, the study focused on the bioactive ions it releases. Gradient dilution experiments determined that a 1 / 8 concentration of ZnCS extract had the best proliferative effect. Treatment of senescent BMSCs with this extract simultaneously downregulated the expression of senescence genes and enhanced osteogenic capacity (e.g., Figure 4 (As shown).

[0061] Further, a suspended particle co-culture model was used to simulate the in vivo microenvironment (50 μg / mL was the optimal concentration). Confocal microscopy confirmed that ZnCS particles could enter the cytoplasm of senescent BMSCs via endocytosis and co-localize with lysosomes. This "dual-mode stimulation" of physical contact and ion release exhibited synergistic biological effects: the particle treatment group significantly improved the senescence phenotype and enhanced the expression of multi-stage osteogenic genes (such as...). Figure 5 , 6 (As shown).

[0062] Zinc feldspar scaffolds promote osteoporotic bone regeneration in vivo.

[0063] To investigate the bone regeneration capacity of zinc feldspar (ZnCS) in a pathological microenvironment, this invention conducted in vivo experiments using an ovariectomy (OVX)-induced osteoporosis rabbit model. A lateral femoral condyle defect model was established 8 weeks after OVX surgery, and implanted materials (Sham-Blank, OVX-Blank, OVX-TCP, and OVX-ZnCS) were selected. Micro-CT reconstruction showed that the amount of newly formed bone tissue (green) within the ZnCS scaffold was significantly better than that in the OVX-Blank and OVX-TCP groups (e.g., ...). Figure 7 As shown in Figure A), quantitative analysis confirmed that the BMD, BV / TV, and trabecular bone thickness (Tb.Th) of newly formed bone in the OVX-ZnCS group were significantly improved (as shown in Figure A). Figure 7(As shown in B, 7C, and 7D). Mechanistic studies have shown that, in terms of anti-aging, the fluorescence intensity of p16INK4A protein in bone tissue of the ZnCS group was significantly lower than that of the control group (e.g., ...). Figure 8 As shown in A and 8C); in terms of promoting bone formation, RUNX2 expression levels were significantly increased (e.g., ...). Figure 8 (As shown in B and 8D). The above results clearly confirm that the ZnCS scaffold significantly enhances the regenerative capacity of osteoporotic bone defects by synergistically clearing senescent cells and activating osteogenic function.

[0064] Oral administration of zinc flavonoid dispersion effectively alleviates bone loss in ovariectomized mice.

[0065] To address the common problems of low absorption and significant side effects of oral supplements (such as calcium tablets and vitamin D) in systemic interventions for osteoporosis, this invention, based on the dual anti-aging and bone-inducing properties of ZnCS demonstrated in in vitro and in vivo experiments, innovatively employs an oral administration strategy to treat an osteoporotic rat model (divided into Sham, OVX, and OVX@ZnCS groups). Micro-CT analysis shows that the bone loss rate in the OVX@ZnCS group was significantly lower than that in the OVX group (e.g., Sham, OVX@ZnCS group). Figure 9 As shown in Figure A), key parameters such as bone volume fraction (BV / TV), trabecular bone number (Tb.N), and trabecular bone spacing (Tb.Sp) significantly improved at week 8 (e.g.). Figure 9 (As shown in B). Immunofluorescence of bone sections showed that oral ZnCS significantly inhibited the accumulation of the bone tissue aging marker p16INK4A (as shown in B). Figure 10 As shown in A and 10C), it also promotes the upregulation of osteogenic-related protein RUNX2 expression ( Figure 10 (As shown in B, 10D).

[0066] In summary, this invention reveals for the first time the anti-aging properties of silicate bioceramics and proposes the innovative concept of "Silicate Anti-Aging (SAS)". Zinc feldspar (ZnCS), in three forms—porous scaffold, ion extract, and particulate suspension—exhibited dual bioactivity in in vitro experiments, demonstrating both delayed cell aging and promotion of osteogenic regeneration. Compared to traditional β-TCP bioceramics, ZnCS can more effectively remodel the aging microenvironment and significantly accelerate the repair of osteoporotic bone defects. Notably, oral administration of ZnCS can systematically inhibit osteoporosis-related bone loss. This silicate bioceramic successfully integrates anti-aging and bone-inducing functions, not only expanding the application boundaries of silicon-based materials but also enriching the theoretical framework of bioceramics.

Claims

1. The application of a silicate bioactive ceramic material with both anti-aging and osteopromoting properties, characterized in that, The silicate bioactive ceramic material is zinc feldspar, with the chemical composition Ca2ZnSi2O7; the zinc feldspar material is in the form of a zinc feldspar scaffold, a zinc feldspar extract, or a zinc feldspar dispersion; the silicate bioactive ceramic material continuously releases Zn... 2+ The zinc feldspar scaffold is used to synergistically activate the SIRT1-mediated PI3K-AKT signaling pathway with SiO32- to inhibit cell senescence and promote bone tissue regeneration; wherein, the zinc feldspar scaffold is used to prepare implantable materials for the regeneration of senescent tissues; and the zinc feldspar extract or zinc feldspar dispersion is used to prepare an oral formulation with dual anti-aging and osteopromoting effects.

2. The application according to claim 1, characterized in that, The silicate bioactive ceramic material releases Zn 2+ The concentration of is 0.3–12.5 μg / mL, and the concentration of SiO32- is 5.0–50.0 μg / mL.

3. The application according to claim 1 or 2, characterized in that, The zinc feldspar material is in the form of a zinc feldspar support; the macroscopic morphology of the zinc feldspar support is a multi-layered mesh support formed by multiple base columns stacked at 45°; preferably, the diameter of a single base column in the zinc feldspar support is 0.4-0.6 mm; the distance between the base columns is 0.8-1.2 mm; The zinc feldspar support has a three-dimensional interconnected pore structure; preferably, the pore size of the three-dimensional interconnected pore structure is 200-800 μm and the porosity is 30-70%.

4. The application according to claim 3, characterized in that, The preparation method of the zinc feldspar scaffold includes: (1) Mix zinc feldspar powder with photosensitive resin to obtain photocurable printing paste; (2) Zinc feldspar scaffold blanks were prepared by photopolymerization printing technology; (3) The obtained zinc feldspar support blank is sintered to obtain the zinc feldspar support.

5. The application according to claim 4, characterized in that, The mass ratio of zinc feldspar powder to photosensitive resin is (40-50):55; The solid content of the photocurable printing paste is 40-50 wt.%.

6. The application according to claim 4 or 5, characterized in that, The sintering temperature is 1250–1270℃, and the holding time is 3–3.5 h.

7. The application according to claim 1 or 2, characterized in that, The zinc feldspar material is in the form of zinc feldspar extract; the preparation method of the zinc feldspar extract includes: mixing zinc feldspar powder with basic culture medium at a ratio of 200 mg / mL to obtain a mixture; after shaking and centrifuging the mixture, collecting the supernatant and sterilizing it to obtain the zinc feldspar extract.

8. The application according to claim 7, characterized in that, The particle size of the zinc feldspar powder is 8.0–70 μm; the basal culture medium is MEM-α medium.

9. The application according to claim 1 or 2, characterized in that, The zinc feldspar material is in the form of a zinc feldspar dispersion; the preparation method of the zinc feldspar dispersion includes: mixing sterilized zinc feldspar powder with a basic culture medium at a ratio of 25-500 μg / mL to obtain the zinc feldspar dispersion.

10. The application according to claim 9, characterized in that, The zinc feldspar powder is disinfected by ultraviolet light for 12–48 hours.

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